Natural and synthetic estrogens are typically considered as "endocrine disruptors" by virtue of their ability to mimic or antagonixe the normal activity of hormone. Although the most well characterized effects of endocrine disruptors are receptor-mediated, non-receptor mediated effects may also be operative in estrogen carcinogenesis as suggested by our most recent demonstration that DES can induce aberrant hypermethylation of a reporter transgene in a novel bioassay. Based on the knowledge that altered DNA methylation is now known to be a driving force in the development of both somatic and inherited cancers, this data provide a strong rationale for pursuing the mechanism by which estrogens and other epigenetic carcinogens can alter DNA methylation. The Epigene assay is a novel mammalian cell mutagenesis assay that exploits a unique set of transgenic Chinese hamster cell lines to distinguish between mutagenic and epigenetic mechanisms of gene inactivation. The short term gola of this project are to evaluate the DNA methylation altering capacity of DES, estradiol, and a gew catechol estrogen metabolites, and X-rays (aim 1); to determine the dose dependence (aim 2) and permanence (aim 3) of any altered DNA methylation that may be induced by these carcinogens. In aim 4, mammary epithelial cells will be employed to screen for genes whose expression may be down regulated by altered DNA methylation following exposure to these carcinogens. The long term goals of this project are to further validate the use of the Epigene Assay for routine experimental and risk assessment used, as well as for mechanistic studies of altered DNA methylation and gene expression.